Hemostatic compositions and methods of making thereof

ABSTRACT

The present invention is directed to hemostatic compositions comprising at least partially integrated agglomerated ORC fibers, fibrinogen, and thrombin and methods of forming a powdered hemostatic composition, comprising the steps of: forming a suspension of a mixture comprising particles of fibrinogen, thrombin, ORC fibers in a non-aqueous low boiling solvent, agitating and shearing said suspension in a high shear mixing reactor, adding water to allow particles to agglomerate, allowing the non-aqueous solvent to evaporate, drying and sieving the composition; and thus forming the powdered hemostatic composition.

FIELD OF THE INVENTION

The present invention relates generally to agents and materials forpromoting hemostasis and tissue sealing and, more particularly, toresorbable hemostatic particulates with improved efficacy, particularlyparticulate aggregates made of fibrinogen, thrombin, and oxidizedregenerated cellulose, and to methods for manufacturing suchcompositions.

BACKGROUND

In a wide variety of circumstances, animals, including humans, cansuffer from bleeding due to wounds or during surgical procedures. Insome circumstances, the bleeding is relatively minor, and normal bloodclotting functions in addition to the application of simple first aidare all that is required. In other circumstances substantial bleedingcan occur. These situations usually require specialized equipment andmaterials as well as personnel trained to administer appropriate aid.

Bleeding during surgical procedures may manifest in many forms. It canbe discrete or diffuse from a large surface area. It can be from largeor small vessels, arterial (high pressure) or venous (low pressure) ofhigh or low volume. It may be easily accessible or it may originate fromdifficult to access sites. The control of bleeding is essential andcritical in surgical procedures to minimize blood loss, to reducepost-surgical complications, and to shorten the duration of the surgeryin the operating room. The selection of appropriate methods or productsfor the control of bleeding is dependent upon many factors, whichinclude but are not limited to bleeding severity, anatomical location ofthe source and the proximity of adjacent critical structures, whetherthe bleeding is from a discrete source or from a broader surface area,visibility and precise identification of the source and access to thesource.

Conventional methods to achieve hemostasis include use of surgicaltechniques, sutures, ligatures or clips, and energy-based coagulation orcauterization. When these conventional measures are ineffective orimpractical, adjunctive hemostasis techniques and products are typicallyutilized.

To address the above-described problems, materials have been developedfor controlling excessive bleeding or as adjuncts to hemostasis. TopicalAbsorbable Hemostats (TAHs) are widely used in surgical applications.TAHs encompass products in various forms, such as based on woven ornon-woven fabrics or sponges, and are typically made of at leastpartially resorbable materials, ranging from natural to syntheticpolymers and combinations thereof, including lactide-glycolide basedco-polymers such as polyglactin 910, oxidized cellulose, oxidizedregenerated cellulose (ORC), gelatin, collagen, chitin, chitosan, starchetc. Gelatin is used in various forms with or without a topical thrombinsolution. Also, widely used are biologically active topical hemostaticproducts (topical thrombin solutions, fibrin sealants, etc.) and avariety of synthetic topical sealants.

To improve the hemostatic performance, scaffolds based on the abovementioned TAH materials can be combined with biologically-derivedclotting factors, such as thrombin and fibrinogen.

Due to its biodegradability and its bactericidal and hemostaticproperties, oxidized cellulose, as well as oxidized regeneratedcellulose has long been used as a topical hemostatic wound dressing in avariety of surgical procedures, including neurosurgery, abdominalsurgery, cardiovascular surgery, thoracic surgery, head and necksurgery, pelvic surgery and skin and subcutaneous tissue procedures.Many methods for forming various types of hemostats based on oxidizedcellulose materials are known, whether made in powder, woven, non-woven,knit, and other forms. Currently utilized hemostatic wound dressingsinclude knitted or non-woven fabrics comprising oxidized regeneratedcellulose (ORC), which is oxidized cellulose with increased homogeneityof the cellulose fiber.

The ORC was introduced in 1960s offering safe and effective hemostasisfor many surgical procedures. The mechanism of action for ORC hemostatsis believed to start with the material absorbing water and then swellingslightly to provide tamponade at the bleeding site. The ORC fibersinitially entrap fluid, blood proteins, platelets and cells forming agel-like “pseudo-clot” which acts as a barrier to blood flow, andsubsequently as a matrix for solid fibrin clot formation. The ORC fabrichas a loose knit in its matrix structure and conforms rapidly to itsimmediate surroundings and easier to manage than other absorbable agentsbecause it does not stick to surgical instruments and its size can beeasily trimmed. This allows the surgeon to hold the cellulose firmly inplace until all bleeding stops.

One of the most commonly used topical hemostatic agents is SURGICEL®Original Absorbable Hemostat, made of an Oxidized Regenerated Cellulose(ORC). SURGICEL® Absorbable Hemostat is used adjunctively in surgicalprocedures to assist in the control of capillary, venous, and smallarterial hemorrhage when ligation or other conventional methods ofcontrol are impractical or ineffective. The SURGICEL® Family ofAbsorbable Hemostats consists of four main product groups, with allhemostatic wound dressings commercially available from Johnson & JohnsonWound Management Worldwide, a division of Ethicon, Inc., Somerville,N.J., a Johnson & Johnson Company:

SURGICEL® Original Hemostat is a white fabric is with a pale-yellow castand has a faint, caramel like aroma. It is strong and can be sutured orcut without fraying.

SURGICEL® NU-KNIT® Absorbable Hemostat is similar but has a denser knitand thus a higher tensile strength. It is particularly recommended foruse in trauma and transplant surgery as it can be wrapped or sutured inplace to control bleeding.

The SURGICEL® FIBRILLAR™ Absorbable Hemostat form of the product has alayered structure which allows the surgeon to peel off and grasp withforceps any amount of SURGICEL® FIBRILLAR™ Hemostat needed to achievehemostasis at a particular bleeding site. The SURGICEL® FIBRILLAR™Hemostat form may be more convenient than the knitted form for hard toreach or irregularly shaped bleeding sites. It is particularlyrecommended for use in orthopedic/spine and neurological surgery.

The SURGICEL® SNoW™ Absorbable Hemostat form of the product is aStructured Non-Woven fabric. SURGICEL® SNoW™ Hemostat may be moreconvenient than other forms of SURGICEL® for endoscopic use due to theStructured Non-Woven fabric. It is highly adaptable and recommended inboth open and minimally invasive procedures.

Other examples of commercial resorbable hemostats containing oxidizedcellulose include GelitaCel® resorbable cellulose surgical dressing fromGelita Medical BV, Amsterdam, The Netherlands. The commerciallyavailable oxidized cellulose hemostats noted above are knitted ornonwoven fabrics having a porous structure for providing hemostasis.

Fibrinogen and thrombin are critical proteins involved in achievinghemostasis after vascular injury and essential to blood clot formation.Fibrinogen and thrombin can be combined in powder form or in anon-aqueous suspension, without initiating a typical clotting reaction,thus preventing the formation of a fibrin clot until the proteins arehydrated in an aqueous medium or other liquid environment in which theproteins are soluble. An admixture of these proteins in powder form havea variety of potential biomedical applications including topicalhemostasis, tissue repair, drug delivery, etc. In addition, an admixtureof these proteins may be loaded onto a carrier or substrate, or othermedical device, in powder form to form a product that may be used forexample as a hemostatic device.

Fibrin sealants, also known as fibrin glue, have been in use in theclinic for decades. Oftentimes, fibrin sealant consists of two liquidcomponents, a fibrinogen comprising component and a thrombin comprisingcomponent, which are stored frozen due to their inherent instability.Sometimes fibrin sealant products consist of two freeze driedcomponents, which require reconstitution immediately prior to use anddelivery by a conjoined syringe or other double-barreled deliverydevice. Freeze dried formulations are typically stable, but thefibrinogen component is difficult to reconstitute. Many hemostaticformulations currently available on the market or in development utilizelyophilized fibrinogen, frequently in combination with lyophilizedthrombin, with hemostatic formulations applied in the form of drypowder, semi-liquid paste, liquid formulation, or optionally disposed ona supporting scaffold such as absorbable fabric scaffold.

To provide dressings with enhanced hemostatic and tissue sealing andadhering properties, therapeutic agents, including, but not limited to,thrombin, fibrin and fibrinogen have been combined with dressingcarriers or substrates, including gelatin-based carriers,polysaccharide-based carriers, glycolic acid or lactic acid-basedcarriers and a collagen matrix. Examples of such dressings are disclosedin U.S. Pat. No. 6,762,336 Hemostatic sandwich bandage, U.S. Pat. No.6,733,774 Carrier with solid fibrinogen and solid thrombin, PCTpublication WO2004/064878 Hemostatic Materials, and European PatentEP1809343B1 A reinforced absorbable multilayered hemostatic wounddressing and method of making.

European Patent No. EP1493451B1 “Haemostatic devices and compositionscomprising oxidized cellulose particles and a polysaccharide binder”discloses that it is problematic to use the carboxylic-oxidizedcellulose as a carrier for acid-sensitive species, such as thrombin andfibrinogen, as well as other acid-sensitive biologics and pharmaceuticalagents. It further discloses a haemostatic composition, consisting of:biocompatible, oxidized cellulose particles having an average designatednominal particle size of from 0.035 to 4.35 mm; a biocompatible, porous,water-soluble polysaccharide binder component other than chitosan; andoptionally, a haemostatic agent selected from thrombin, fibrinogen orfibrin, wherein the weight ratio of said water-soluble polysaccharide tosaid oxidized cellulose particles is from 3:97 to 15:85, and whereinsaid composition is a porous foam sponge obtainable by a processcomprising the steps of: providing a polymer solution having saidpolysaccharide binder component dissolved in a suitable solvent,providing said biocompatible, oxidized cellulose particles, contactingsaid polymer solution with said oxidized cellulose particles underconditions effective to disperse said oxidized cellulose particlessubstantially homogenously throughout said polymer solution to form asubstantially homogenous dispersion, subjecting said polymer solutionhaving said particles dispersed throughout to conditions effective tosolidify said substantially homogenous dispersion; and removing saidsolvent from the solidified dispersion, thereby forming said haemostaticcomposition.

Russian patent publication RU2235539C1 “Method for preparing powder-likematerial for cessation bleeding” discloses method for preparingpowder-like material eliciting hemostatic effect involves mixingpartially oxidized cellulose as a base in an aqueous medium withthrombin and fibrinogen. Gelatin, epsilon-aminocaproic acid and lysozymeare added to indicated substances additionally, and dialdehyde celluloseas fabric is used as partially oxidized cellulose, i.e the content ofaldehyde groups is from 4 to 6% in the following ratio of components:dialdehyde cellulose, 1 g; fibrinogen, 18-22 mg; gelatin, 27-33 mg;epsilon-aminocaproic acid, 45-55 mg; lysozyme, 9.5-10.5 mg; thrombin,350 U; water, 6.5 ml. Method involves preparing solution containingfibrinogen, epsilon-aminocaproic acid in one-half amount of totalcontent of gelatin and one-half amount of total content of water, andseparated preparing solution of thrombin, lysozyme and remained amountof gelatin in remained amount of water. In prepared solutions, one-halfamount of dialdehyde cellulose is kept for 3-4 h, semi-finished productsare squeezed, dried in air and subjected for the combined grinding.

U.S. patent publication No. 20060159733A1 “Method of providinghemostasis to a wound” discloses that the acidic nature of carboxylicoxidized cellulose substrate could rapidly denature and inactivate acidsensitive proteins, including thrombin or fibrinogen, on contact. Muchof the enzymatic activity of thrombin and Factor XIII could be lostduring the reaction. This makes it difficult to use thecarboxylic-oxidized cellulose as a carrier for thrombin, fibrinogen,fibrin, or other acid sensitive biologics and pharmaceutical agents. Itfurther discloses that hemostatic wound dressings containing neutralizedcarboxylic-oxidized cellulose and protein based-hemostatic agents, suchas thrombin, fibrinogen and fibrin are known. Neutralizedcarboxylic-oxidized cellulosic materials are prepared by treating theacidic carboxylic-oxidized cellulose with a water or alcohol solution ofa basic salt of a weak organic acid to elevate the pH of the cellulosicmaterial to between 5 and 8 by neutralizing the acid groups on thecellulose prior to addition of thrombin to make it thrombin compatible.A thrombin hemostatic patch was disclosed, wherein thrombin was added toan acidic carboxylic oxidized regenerated cellulose or other material inpresence of an acid neutralizing agent, epsilon aminocaproic acid(EACA), to raise the pH of the material to a region where thrombin canperform as a hemostat. While such neutralized carboxylic-oxidizedcellulose may be thrombin compatible, it is no longer bactericidal,because the anti-microbial activity of oxidized cellulose is due to itsacidic nature.

U.S. Pat. No. 7,094,428B2 “Hemostatic compositions, devices and methods”discloses a hemostatic composition which comprises at least oneprocoagulant metal ion, such as silver (I) or mercury (II), and at leastone procoagulant biopolymer, such as collagen, thrombin, prothrombin,fibrin, fibrinogen, heparinase, Factor VIIa, Factor VIII, Factor IXa,Factor Xa, Factor XII, von Willebrand Factor, a selectin, a procoagulantvenom, a plasminogen activator inhibitor, glycoprotein IIb-IIIc, aprotease, or plasma. The composition in the form of a paste, dough,glue, liquid, lyophilized powder or foam, may be provided, forapplication to a wound. A hemostatic composition comprising at least oneprocoagulant biopolymer in combination with a procoagulant metal ion,said procoagulant metal ion present in said composition at a level belowits effective hemostatic concentration in the absence of saidprocoagulant biopolymer wherein the hemostatic composition is selectedfrom the group consisting of silver (I) and collagen, silver (I) andthrombin, silver (I) and prothrombin, silver (I) and fibrin, silver (I)and fibrinogen, silver (I) and heparinase, silver (I) and Factor VIIa,silver (I) and Factor VIII, silver (I) and Factor IXa, silver (I) andFactor Xa, silver (I) and Factor XII, silver (I) and von WillebrandFactor, silver (I) and a selectin, silver (I) and a procoagulant venom,silver (I) and a plasminogen activator inhibitor, silver (I) andglycoprotein IIb-IIIa, silver (I) and a protease, silver (I) and plasma,mercury (II) and collagen, mercury (II) and thrombin, mercury (II) andprothrombin, mercury (II) and fibrin, mercury (II) and fibrinogen,mercury (II) and heparinase, mercury (II) and Factor VIIa, mercury (II)and Factor VIII, mercury (II) and Factor IXa, mercury (II) and FactorXa, mercury (II) and Factor XII, mercury (II) and von Willebrand Factor,mercury (II) and a selectin, mercury (II) and a procoagulant venom,mercury (II) and a plasminogen activator inhibitor, mercury (II) andglycoprotein IIb-IIIa, mercury (II) and a protease, and mercury (II) andplasma. The hemostatic composition of the invention may also include acarrier, such as, but not limited to, polyethylene glycol, hyaluronicacid, cellulose, oxidized cellulose, methyl cellulose, or albumin. Thesemay be used to provide a matrix, a suitable viscosity, deliverability,adherence, or other properties desired to be imparted to thecompositions herein for easy in application to a wound. Numerous othercarrier which impart these characteristics are embraced herein.

U.S. Pat. No. 6,162,241A “Hemostatic tissue sealants” discloses ahemostatic tissue sealant, comprising: a biocompatible, biodegradablehydrogel tissue sealant comprising crosslinkable groups havingincorporated therein an effective amount of a hemostatic agent to stopthe flow of blood from tissue in a medically acceptable period of time.

U.S. Pat. No. 6,177,126B1 “Process for the production of a material forsealing and healing wounds” discloses a process for the production of amaterial for sealing and/or healing wounds, comprising: i) filling aliquid composition into a container having two or more plates, at leasttwo of said plates being perforated with one or more flow-through holesand at least one of said perforated plates being movable relative toanother of said perforated plates, ii) transporting a carrier below thecontainer in a transport direction, and iii) continuously moving theperforated plates relative to each other so as to allow the liquidcomposition to drip on to the carrier being transported below thecontainer, whereby the liquid composition is substantially evenlyapplied to the carrier.

PCT publication No. WO2014135689A2 “Powder formulation” discloses asterile powder composition suitable for medical use comprising thrombinand fibrinogen, wherein the thrombin powder is produced from a liquidfeedstock, wherein the feedstock comprises a solution or a suspension ofthrombin, preferably a solution, wherein the powder is produced byremoval of liquid by a process selected from aseptic spray drying oraseptic fluid bed drying, and wherein the powder resulting from removalof liquid from the feedstock exhibits at least 80% of the thrombinpotency or activity of the liquid feedstock, and wherein the fibrinogenpowder is produced by removal of liquid from a feedstock, wherein thefeedstock comprises a solution or a suspension of fibrinogen, preferablya solution, by aseptic spray drying or aseptic fluid bed drying, andwherein said composition is packaged as a sterile final pharmaceuticalproduct for medical use.

U.S. Patent Publication No: 2010/0119563 “SOLID FIBRINOGEN PREPARATION”discloses a solid fibrinogen preparation comprising fibrinogen andfurther comprising: (a) albumin; (b) a nonionic surfactant; (c) a basicamino acid or a salt thereof; and (d) at least two amino acids or a saltthereof selected from the group consisting of an acidic amino acid, asalt thereof, a neutral amino acid and a salt thereof.

Additional patent documents related to formulating hemostaticformulations include:

U.S. Pat. No. 9,717,821B2 Formulations for wound therapy

U.S. Pat. No. 7,052,713B2 Carrier with solid fibrinogen and solidthrombin

U.S. Pat. No. 9,724,213B2 Nanocrystalline cellulose materials andmethods for their preparation

U.S. Pat. No. 8,840,877B2 Polysaccharide-protein conjugates reversiblycoupled via imine bonds

U.S. Pat. No. 6,200,587B1 Tissue sealant containing fibrinogen, thrombinand carboxymethyl cellulose or salt thereof

U.S. Pat. No. 8,846,105B2 Dry powder fibrin sealant

US20160015792A1 POWDER FORMULATION COMPRISING THROMBIN AND FIBRINOGEN

U.S. Pat. No. 6,596,318B2 Fibrin tissue adhesive formulation and processfor its preparation

KR1624625B1 Improved absorbable hemostatic material and method forpreparing the same

KR1588633B1 Composition and kit for forming gel for hemostasis andadhesion inhibition|

KR804434B1 Fibrin-based glue granulate and corresponding productionmethod|Fibrin-based glue granulate and a method of manufacturing thesame.

U.S. Pat. No. 9,795,773B2 Medicament unit dose cartridge and deliverydevice

U.S. Pat. No. 7,351,422B2 Hemostatic soluble cellulose fibers containingcoagulating protein for treating wound and process for producing thesame

CN101716383A Preparation method of bleeding stopping and adherencepreventing material|Preparation method for stanching and anti-blockingmaterial

U.S. Pat. No. 5,484,913A Calcium-modified oxidized cellulose hemostat

EP918548B1 USE OF OXIDIZED CELLULOSE AND COMPLEXES THEREOF FOR CHRONICWOUND HEALING

There is a need in improved hemostatic forms and materials whichfacilitate ease of application and rapid onset of hemostasis.

SUMMARY OF THE INVENTION

The present invention is directed to a hemostatic material comprisingaggregates comprising fibrinogen, thrombin, and oxidized regeneratedcellulosic fibers. In some aspects, the hemostatic material furtherincludes additives, such as calcium chloride, Tris. In another aspect,the present invention is directed to a method of making the hemostaticmaterials described above by suspending a mixture of fibrinogen,thrombin, and ORC powders in a non-aqueous solvent, spraying thesuspension through a nozzle onto a substrate, removing the hemostaticmaterial from the substrate and sieving.

In yet another aspect, the present invention is directed to a method oftreating a wound by applying the hemostatic materials described aboveonto and/or into the wound of a patient.

In one embodiment, the present invention relates to methods of forming apowdered hemostatic composition by forming a suspension of a mixturecomprising particles of fibrinogen, thrombin, ORC fibers in anon-aqueous low boiling solvent; spraying the suspension through anozzle onto a substrate, allowing the non-aqueous solvent to evaporate;separating the composition from the substrate and sieving thecomposition; and thus, forming the powdered hemostatic composition. Thenon-aqueous low boiling solvent can be hydrofluoroether C₄F₉OCH₃, suchas but not limited to HFE7100. The suspension can further include Trisand/or calcium chloride. The liquid suspension can contain a fibrinsealant powder which comprises about 90% of fibrinogen, about 8% ofthrombin, and about 2.5% calcium chloride by weight. The suspendedpowdered hemostatic composition can have a ratio of fibrin sealantpowder to ORC from about 1:1 to about 10:1 by weight. The suspendedpowdered hemostatic composition can be in the form of a powder having ameasured particle size predominantly in the range from about 250 toabout 850 microns, more preferably from about 355 to about 850 microns.The resulting powdered hemostatic composition comprises at leastpartially integrated agglomerated ORC fibers, fibrinogen, and thrombinand can further comprise Tris and/or calcium chloride.

The present invention is further directed to methods of treating a woundby applying the resulting hemostatic composition described above ontoand/or into the wound.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic diagram of the manufacturing process.

FIG. 2 is a photo showing test vials evaluating gelling of inventive andcomparative compositions in water.

FIG. 3 is a photo showing test vials evaluating clotting of blood incontact with inventive and comparative compositions.

FIG. 4 is a photo showing test vials evaluating clotting of blood incontact with inventive and comparative compositions.

FIG. 5 is a composite photo showing the results of solubilizationtesting of the comparative compositions.

FIG. 6 is a composite photo showing the results of solubilizationtesting of the inventive compositions.

FIG. 7 is a composite photo showing the results of solubilizationtesting of the comparative compositions.

FIG. 8 is a composite photo showing the results of solubilizationtesting of the inventive compositions at varying particle size.

FIG. 9 is showing SEM images of one of inventive compositions.

FIG. 10 is a schematic diagram of the HSM manufacturing process.

FIG. 11 is a schematic diagram of the HSM manufacturing reactor.

FIG. 12 shows inventive hemostatic powder prepared by the HSM Method ofExample 9.

FIG. 13 shows a Chart of gel strength in kPa.

FIG. 14 shows the results of testing in animal model of the hemostaticpowders made by using different solvents/binders.

FIG. 15 shows schematic block-diagram of the HSM manufacturing processfor preparation of hemostatic powders using acetone and polymer RG502.

FIG. 16 shows schematic block-diagram of the HSM manufacturing processfor preparation of hemostatic powders using acetone and water.

FIG. 17 shows schematic block-diagram of the HSM manufacturing processfor preparation of hemostatic powders using 96% ethanol.

FIG. 18 shows schematic block-diagram of the HSM manufacturing processfor preparation of hemostatic powders using HFE/water.

FIG. 19 shows density properties of hemostatic powders obtained withvarious solvents/binders.

FIG. 20 shows gel strength properties of hemostatic powders obtainedwith various solvents/binders.

FIG. 21 shows Lung leak model with 1.2 cm long incision.

FIG. 22 shows the inventive hemostatic powder forming a gel (counted for2 min) on the deflated lung.

FIG. 23 shows the inventive hemostatic powder forming a gel that sealsthe lung leak at 30 cm H₂O.

FIG. 24 shows Lung leak model with 1 cm long incision.

FIG. 25 shows the inventive hemostatic powder forming a gel that sealsthe lung leak at 30 cm H₂O.

FIG. 26 shows the inventive hemostatic powder forming a gel partiallydelaminated at 40 cm H₂O pressure (arrow indicates the delaminationarea).

DETAILED DESCRIPTION

The inventors have discovered hemostatic materials and process formaking thereof, the hemostatic materials having surprising and highlybeneficial properties for hemostasis.

The hemostatic material according to the present invention is made fromoxidized cellulose-based fiber materials, more preferably form oxidizedregenerated cellulose powder, fibrinogen powder, and thrombin powder.The hemostatic material according to the present invention represents atleast partially integrated ORC fibers, fibrinogen, and thrombin in aform of a powder.

Referring to FIG. 1, a schematic block-diagram of the process of makingthe hemostatic material according to the present invention is shown andcomprises the steps of:

-   -   Preparing dry powders of fibrinogen, thrombin, and ORC    -   Suspending a mixture of fibrinogen, thrombin, and ORC powders in        a non-aqueous solvent capable of rapid evaporation under ambient        conditions    -   Spraying the suspension through a nozzle onto a substrate    -   Allowing the non-aqueous solvent to evaporate and drying the        resulting hemostatic material    -   Removing/separating the hemostatic material from the substrate        and sieving, thus forming at least partially integrated ORC        fibers, fibrinogen, and thrombin in a form of a powder

In one embodiment, Tris, or Tris(hydroxymethyl)aminomethane buffer in apowder form is added to the fibrinogen, thrombin, and ORC mixture for pHadjustment. Then the mixed composition was added to HFE to form asuspension. In one embodiment, the cooling/chilling effect of thematerial stream during spraying due to HFE evaporation allows someambient moisture to be absorbed onto or into the resulting hemostaticmaterial. Any excessive moisture thus absorbed is removed in the finaldrying step in vacuum oven drying.

According to one aspect of the present invention, the ratio offibrinogen/thrombin mixture to ORC powder in the inventive hemostaticmaterial is from about 1:1 to about 10:1 by weight.

According to one aspect of the present invention the inventivehemostatic material comprises particles having size of 250-850 microns.

According to one aspect of the present invention the inventivehemostatic material comprises substantially uniformly distributed atleast partially integrated ORC fibers, fibrinogen, and thrombin in aform of a powder

According to one aspect of the present invention, the inventivehemostatic material has high uniformity, integration, fastgelling/clotting, and strong adhesion force.

According to one aspect of the present invention, the collecting surfaceor substrate onto which the suspension is sprayed, comprises an inertnon-woven felt or mesh or a steel plate.

According to another embodiment of the present invention, the presentinvention is directed to a hemostatic material comprising aggregatescomprising fibrinogen, thrombin, and oxidized regenerated cellulosicfibers. In some aspects, the hemostatic material further includesadditives, such as calcium chloride, and a buffer, such as Tris and/orLysine.

In another aspect, the present invention is directed to a method ofmaking the hemostatic materials described above by suspending a mixtureof fibrinogen, thrombin, and ORC powders in a non-aqueous solvent in aHigh Shear Mixing (HSM) apparatus, and performing high shear mixing andregular mixing and/or agitation, while allowing the volatile non-aqueoussolvent to evaporate through any suitable port(s) such as through airseals of mixing blades/impellers and/or through a vacuum tube.Optionally, a small amount of water or aqueous solution is introducedinto the reactor during mixing to aid particlesbinding/aggregation/clumping. The non-aqueous low boiling solvent can behydrofluoroether C₄F₉OCH₃, such as but not limited to HFE7100.

According to an embodiment of the present invention, a hemostaticaggregated powder comprises ORC fine fibers or powder, thrombin,fibrinogen, lysine (as a buffer/pH adjusting agent), calcium salt.

According to an embodiment of the present invention, a process of makingsuch powdered hemostatic product comprises the steps of mixing theindividual components as powders with HFE, creating a suspension in HFE(volatile non-aqueous solvent) in High Shear mixing/shearing reactor,having a low speed mixing blade and high-speed shear blade. As mixing isperformed, HFE can evaporate through air seals of mixing blades and/orvacuum tube. A small amount of water is introduced into the reactor toaid particles binding/aggregation/clumping.

According to an embodiment of the present invention, a method of makinghemostatic powder, comprising the steps of: Forming a suspension of ORCpowder, thrombin, fibrinogen, lysine, calcium salt in HFE (volatilenon-aqueous solvent), agitating the suspension with high speed shearblade (and optionally continuously mixing the components simultaneouslywith a low speed mixer); Adding a small amount of water to the reactorto aid particles binding/aggregation/clumping; allowing HFE to evaporatefrom the reactor completely; Thus forming the hemostatic powder.

Example 1. Preparation of Hemostatic Compositions

The individual components of the hemostatic compositions of the presentinvention were prepared as described below.

Fibrinogen. Any method of preparation of fibrinogen powder can beutilized, including lyophilization, freeze drying, etc. In the instantexample, fibrinogen powder was prepared by spray drying method (Spraydryer manufacturer: ProCepT, Model: 4M8-TriX). Fibrinogen solution is aformulation commercially available from Bioseal Biotech CO. LTD, locatedin Guangzhou, China, and comprising fibrinogen, albumin, and otherneeded reagents in WFI. The fibrinogen solution was first atomizedthrough a spray nozzle in a hot airflow, then dried instantly. The spraydrying parameters were as shown in Table 1

TABLE 1 Feed Rate 130 ml/h Drying Columns 3 (Mode II) Column Air Flow0.6 m³/min Inlet Air Temperature 150° C. Nozzle Diameter 0.8 mmAtomizing Air Flow 12 L/min Cyclone Gas 0.15 m³/min

Thrombin. Any method of preparation of thrombin powder can be utilized,including lyophilization, freeze drying, etc. In the instant example,thrombin powder was prepared by spray drying method with thrombinformulation solution. Thrombin solution was the formulation commerciallyavailable from Bioseal Biotech CO. LTD, located in Guangzhou, China, andcomprising thrombin, albumin, and other needed reagents in WFI. Thespray drying parameters were as shown in Table 2

TABLE 2 Feed Rate 258 ± 20 ml/h Drying Columns 2 Column Air Flow 0.3m³/min Cooling Air Flow 0.3 m³/min Inlet Air Temperature 160° C. NozzleDiameter 0.4 mm Atomizing Air Flow 7 L/min Cyclone Gas 0.1 m³/min

The thrombin and fibrinogen powders were then mixed together forpreparation of the composite by the ratio of 89.7% of fibrinogen 7.8% ofthrombin and 2.5% calcium chloride by weight, thus forming fibrinsealant powder.

The source of fibrinogen and thrombin was Porcine blood plasma which wasfractionated to obtain fibrinogen and thrombin. and supplied by BiosealBiotech CO. LTD, Located in Guangzhou, China

The ORC powder can be obtained by processing of the Surgicel originalfabric. A reference is made to the U.S. Provisional Patent ApplicationNo. 62/251,773 by Yi-Lan Wang, filed 6 Nov. 2015 and titled “CompactedHemostatic Cellulosic Aggregates”, which is incorporated by reference inits entirety for all purposes.

Briefly, the ORC powder was obtained by processing of the Surgiceloriginal fabric in the following process:

1) split and cut the fabric into about 2″×8″ pieces,2) mill the fabrics into the powder particle size (D50 less than 94microns) using known milling methods. One of the methods used in thepreparations is the ball mill method—place ˜100 grams of fabric into a500-ml zirconia jar, then place 12 to 13 pieces of 20 mm zirconia balls(agates) into the same jar, cover and fix the jar in a Retsch planetaryball mill (Model PM100), mill the fabric with 450 rpm for 20 minutes,transfer the milled powders onto a 8″ diameter and 300-micron openingsieve, separate the agates and the powders by slightly shaking, andcollect the powders.

The inventive hemostatic compositions were prepared as follows by usingco-spraying methods. 1 part of ORC fiber was combined with 1 part, or 2parts, or 5 parts, or 10 parts of fibrin sealant powders, by weight.Thus, as an example, 10 g of ORC powder was combined with 10 g, 20 g, 50g, or 100 g of fibrin sealant mixed powders, to produce 20 g, 30 g, 50g, or 100 g of mixture.

A small amount of Tris was added to adjust pH to 7.0 for each respectedORC: Fibrin sealant ratio. pH was adjusted by placing the powder on awetting surface and measuring the resulting pH and evaluating the amountof Tris needed for obtaining a neutral pH of 7. The sample was thendiscarded. A corresponding proportional amount of dry powder of Tris wasthen added to the powder mixture, prior to co-spraying.

ORC is not per se neutralized as Tris is added in dry form. ORC is beingneutralized when the whole powder formulation is wetted during theapplication and the Tris is dissolved. A low boiling non-aqueous solventwas utilized for making a suspension of FS and ORC powders.Hydrofluoroether C4F9OCH3 was used, obtained as HFE 7100, for instancesupplied by 3M as Novec 7100 Engineered Fluid, having boiling point of61° C. HFE7100 solvent was added to the powders compositions andfiltered through 150 μm sieve. The evenly distributed suspension wascreated by constantly agitating at 90 rpm/min in the reservoir at 20±5°C. temperature. The suspended components were sprayed through a 0.7 mmdiameter nozzle. The type of the nozzle used wasB1/8VAU-316SS+SUV67-316SS manufactured by Spraying Systems Co. The flowthrough the nozzle was at a flow rate of 130 ml/min, onto a non-wovenfabric or stainless steel substrate at 20±5° C. temperature.

Upon spraying the suspension, most of the HFE 7100 solvent wasevaporating. The HFE 7100 solvent residues and the absorbed ambientmoisture in the powder, if any, could evaporate in a vacuum drying ovenfor 24 h±5 h at 20±5° C.

The resulting hemostatic composition was scooped off or peeled off orotherwise separated from the substrate, and sequentially passed throughsieves having 850 μm, 355 μm, 250 μm openings.

Table 3 shows parameters used for co-spraying compositions

TABLE 3 PARAMETERS FOR CO-SPRAYING THE COMPOSITION -fan pressure 0.3 baratomizing pressure 3 bar flow rate 130 ml/min liquid pressure 16 kpaNozzle Diameter 0.7 mm agitation speed 90 rpm/min

As comparative examples, pure fibrin sealant mixture composition with noORC and no Tris was also prepared by the same spray method HFE7100.

Example 2. Comparison of Mechanically Mixed Vs. Spray Mixed HemostaticCompositions Gelling

Rapid gelling and formation of strong gels is important for hemostaticmaterials.

The inventive hemostatic composition was prepared as described above byco-spray method.

The comparative mechanically mixed composition was prepared by manuallyshaking dry powders, i.e. FS and ORC powders obtained as describedabove, by manually shaking in a container into an evenly mixedcomposition, with no co-spraying performed. The compositions includedall the same components, including Tris, with the difference beingmethods of mixing.

Fibrin sealant powder to ORC ratios tested were: Fibrin sealant (FS):ORC ratios—1:1; 2:1; 5:1; 10:1 (by weight). Thus for 1:1 FS/ORC ratio, 1part of fibrin sealant powder (comprising fibrinogen, thrombin, calciumchloride) is combined with 1 part of ORC powder (by weight).

Mechanically mixed composition and the inventive spray mixed compositionwere then added to 20 ml of water in a 50-ml vial in the amount of 200mg on the top of the water surface. After 2 min for gelling, the vialwas turned upside down and observations made if a gelled layer ofcomposition was formed in which case the water was observed sealed bythe gelled layer and was held on the bottom of the vial by the formedgel layer.

Referring now to FIG. 2, showing an image of the test vials turnedupside down at the end of the test, with

-   -   test vial 1 showing mechanically mixed composition 1:1 FS/ORC        ratio    -   test vial 2 showing mechanically mixed composition 2:1 FS/ORC        ratio    -   test vial 3 showing mechanically mixed composition 5:1 FS/ORC        ratio    -   test vial 4 showing mechanically mixed composition 10:1 FS/ORC        ratio    -   test vial 5 showing mechanically mixed composition with no ORC        powder (fibrin sealant powder only)    -   test vial 6 showing inventive hemostatic composition 1:1 FS/ORC        ratio

Analysis of the results shown in FIG. 2 indicates that in test vials 1-5gelling was insufficient to hold the fluid and fluid is visible in thelower part of the vial. In test vial 6, fluid is visible in the upperpart of the vial, i.e. water is being held by the gelled layer andprevented from moving to the lower part of the vial under gravitationalforce. Thus, for mechanically mixed compositions in all ratios and forfibrin sealant composition without ORC gelling was insufficient, whilethe inventive hemostatic composition prepared in 1:1 ratio exhibitedsurprisingly strong gelling.

Example 3. In Vitro Testing of Blood Clotting

In vitro clotting of blood by several inventive and comparativecompositions was tested as follows.

20 ml of citrated whole blood (porcine) was added to a 50-ml vial. 200mg of hemostatic compositions being tested were added on the top of theblood surface. After 2 min for clotting, the vial was turned upside downand observations of blood clotting were made. In case of completeclotting, the clotted blood stays in the upper part of the vial turnedupside down. In case of incomplete clotting, blood remains fluid anddrains towards the lower part of the vial due to the gravitationalforce.

Comparative excipients added to fibrin sealant powder instead of ORCwere Trehalose, PEG4000, Mannitol, and Alfa-cellulose (α-cellulose). Allexcipients were purchased from Aladdin industrial corporation. Fibrinsealant powder mixtures with Trehalose, PEG 4000, Mannitol, orAlfa-cellulose were prepared by spray method as described above in 10:1ratio, i.e. with 10 parts of fibrin sealant (FS) powder combined with 1part of the respective excipient. The total amount of inventive andcomparative compositions added to 20 ml of blood was 200 mg.

Referring now to FIG. 3, showing an image of the test vials turnedupside down at the end of the test, with

-   -   test vial 8 showing the inventive hemostatic composition having        2:1 FS/ORC ratio    -   test vial 9 showing the inventive hemostatic composition having        5:1 FS/ORC ratio    -   test vial 10 showing the inventive hemostatic composition having        1:1 FS/ORC ratio    -   test vial 11 showing comparative composition comprising fibrin        sealant powder only with no ORC made by co-spray    -   test vial 12 showing comparative composition comprising fibrin        sealant powder with addition of α-cellulose in 10:1        FS/α-cellulose ratio by weight    -   test vial 13 showing comparative composition comprising fibrin        sealant powder with addition of trehalose in 10:1 FS/trehalose        ratio by weight    -   test vial 14 showing comparative composition comprising fibrin        sealant powder with addition of PEG4000 in 10:1 FS/PEG4000 ratio        by weight    -   test vial 15 showing comparative composition comprising fibrin        sealant powder with addition of mannitol in 10:1 FS/mannitol        ratio by weight

Analysis of the results presented in FIG. 3 indicates that in test vials8-10 containing the inventive hemostatic composition blood has clotted,with blood clot visible in the upper part of the vial turned upside downwith clotted blood prevented from moving to the lower part of the vialunder gravitational force. Thus, the inventive hemostatic compositionprepared in 2:1; 5:1; 1:1 FS/ORC ratio exhibited surprisingly strongclotting of blood. Also, comparative sample containing α-cellulose invial 12 shows clotting of blood. Comparative examples in vial 11 (fibrinsealant powder only with no ORC); vial 13 (fibrin sealant powder withaddition of trehalose); vial 14 (fibrin sealant powder with addition ofPEG4000); vial 15 (fibrin sealant powder with addition of mannitol) showno clotting or insufficient clotting, whereby clotting was insufficientto hold the fluid and fluid is visible in the lower part of the vial,i.e. blood remains fluid and drains towards the lower part of the vialdue to the gravitational force. The inventive hemostatic compositionsexhibited surprisingly strong blood clotting.

Using the same testing methods, additional in vitro blood clottingtesting was performed for inventive hemostatic composition andcomparative mechanically mixed compositions prepared by manually shakingdry powders in a container as well as for fibrin sealant powder onlywith no ORC

Referring now to FIG. 4, showing an image of the test vials turnedupside down at the end of the test, with

-   -   test vial 1 showing mechanically mixed composition 1:1 FS/ORC        ratio    -   test vial 2 showing mechanically mixed composition 2:1 FS/ORC        ratio    -   test vial 3 showing mechanically mixed composition 5:1 FS/ORC        ratio    -   test vial 4 showing mechanically mixed composition 10:1 FS/ORC        ratio    -   test vial 5 showing comparative composition (200 mg) comprising        compacted ORC powder aggregates prepared as described in the        U.S. Provisional Patent Application No. 62/251,773 by Yi-Lan        Wang, filed 6 Nov. 2015 and titled “Compacted Hemostatic        Cellulosic Aggregates”    -   test vial 6 showing comparative composition comprising fibrin        sealant powder only with no ORC prepared by co-spray method    -   test vial 7 showing the inventive hemostatic composition having        1:1 FS/ORC ratio    -   test vial 8 showing the inventive hemostatic composition having        2:1 FS/ORC ratio    -   test vial 9 showing the inventive hemostatic composition having        5:1 FS/ORC ratio    -   test vial 10 showing the inventive hemostatic composition having        10:1 FS/ORC ratio

Analysis of the results presented in FIG. 4 indicates that comparativeexamples in test vials 1-6, containing mechanically mixed compositionsin all ratios; ORC powder only; and fibrin sealant powder without ORC,show no clotting or insufficient clotting, whereby clotting wasinsufficient to hold the fluid and fluid is visible in the lower part ofthe vial, i.e. blood remains fluid and drains towards the lower part ofthe vial due to the gravitational force. On the contrary, and like theresults presented in FIG. 3, in test vials 7-10, containing theinventive hemostatic composition, the blood has clotted, with blood clotvisible in the upper part of the vial turned upside down with clottedblood prevented from moving to the lower part of the vial undergravitational force. Thus, the inventive hemostatic compositionsprepared in 1:1-10:1 FS/ORC ratios exhibited surprisingly strongclotting of blood.

Example 4. Composition Solubilisation

Rapid solubilisation or solubility of a powdered hemostatic compositionwhen in contact with bodily fluids can help to establish rapidhemostasis and indicates rapid interaction with fluids. The visual testof solubilisation was performed as follows: 1 gram of tested hemostaticpowdered composition was evenly applied to an area of a wettingsubstrate which comprising a non-woven fabric positioned on top of asponge material which was placed in a tray with pure water. After thetested powdered hemostatic composition was applied to the surface of thewetting substrate, visual observation of the composition solubility wasperformed and results recorded at zero time (immediately after applyingthe composition, at 1 min and at 2 min after applying the testedcomposition.

Referring now to FIG. 5, a composite image is shown representing theresults of testing of the comparative mechanically mixed compositionprepared by manually shaking dry powders in a container. The imagestaken at 0, 1, and 2 min for FS/ORC ratios—1:1; 2:1; 5:1; 10:1 as wellas for FS powder with no ORC. The results indicate poor solubilisationeven at 2 min time point for the comparative examples.

Referring now to FIG. 6, a composite image is shown representing theresults of testing of the inventive hemostatic composition prepared bythe spray method. The images taken at 0, 1, and 2 min for FS/ORCratios—1:1; 2:1; 5:1; 10:1 as well as for FS powder with no ORC. Theresults indicate good solubilisation even at 1 min time point and verygood solubilisation at 2 min time point, with rapid full solubilisationobserved for 1:1 and 2:1 ratios already at 1 min and good solubilisationobserved for all ratios at 2 min. Pure FS is showing poor solubilisationeven at 2 min time point for the comparative example.

Referring now to FIG. 7, a composite image is shown representing theresults of testing of the comparative compositions comprising FS withexcipients added to fibrin sealant powder instead of ORC as well as forFS powder with no ORC. The excipients were Trehalose, PEG4000, Mannitol.Fibrin sealant powder mixtures with Trehalose, PEG 4000, Mannitol, wereprepared by spray method as described above in 10:1 FS/excipient ratio.The images taken at 0, 1, and 2 min are shown. The results indicate poorsolubilisation even at 2 min time point for the comparative examples.

The solubility of inventive hemostatic composition prepared by the spraymethod was affected by the concentration of the ORC component. Even atlow concentrations of ORC, the solubility of the composition hasimproved.

Example 5. Particle Size Effects

The effects of the particle size on the performance of the inventivehemostatic compositions were evaluated. Particle size was controlled bysequentially passing the composition through sieves with apertures of850 μm, 355 μm and 250 μm. Inventive composition powders were separatedinto size groups of predominantly above 850 μm, predominantly 355-850μm, predominantly 250-355 μm and predominantly below 250 μm. Inventivehemostatic compositions made with 5:1 FS/ORC ratio were tested forsolubility.

Referring now to FIG. 8, a composite image is shown representing theresults of testing of the inventive hemostatic composition prepared bythe spray method. The images taken at 0, 1, and 2 min for differentpowder size ranges. The results indicate particularly excellentsolubilisation at 2 min point for predominantly 355-850 μm and goodsolubilisation for predominantly 250-355 μm compositions, with lesseffective solubilisation for compositions predominantly above 850 μm,and predominantly below 250 μm. Thus, the range of predominantly 250-850μm is showing good solubilisation and is the preferred range forparticle size, with particles predominantly in the 355-850 μm rangeparticularly preferred. The resulting powder is an agglomerate offibrinogen, thrombin, and ORC, and has many particles with size largerthan the starting materials particle size.

Example 6. Effects of Tris Addition

A peel test of the inventive compositions with added Tris and withoutadded Tris was performed. Tris additions were titrated to achieve pH=7.Tris powder was ground and passed through a 150 μm sieve. The powder ofbelow 150 μm was collected and added the pre-determined amount into thedry mixture to adjust the pH of the composition, prior to co-spray.

The peel test was performed as follows. 0.5 g of the Inventivecomposition was applied to the corium tissue, covered by a compositebi-layer matrix which was pressed into the powder for 3 minutes, thesample-from-tissue separation force was measured by an Instron tensiletesting machine and recorded as force per unit width (N/m). Thecomposite bi-layer matrix comprised a layer of synthetic absorbable poly(glycolide-co-lactide) (PGL, 90/10 mol/mol) nonwoven fabricneedlepunched into a knitted carboxylic-oxidized regenerated cellulosefabric (ORC), as described in U.S. Pat. No. 7,666,803 by D. Shetty etal., titled “Reinforced absorbable multilayered fabric for use inmedical devices”, which is incorporated by reference herein.

Referring now to Table NN, adhesion forces of inventive formulations areshown as a function of ORC addition. While the adhesion is lower athigher ORC content even 1:1 FS powder: ORC fiber formulation hasappreciable peel force.

Referring now to Table 4, adhesion Forces of inventive formulations withand without Tris are shown for different FS/ORC ratios along withcorresponding pH values. Tris was added in weight percentages listed toadjust pH to 7.0. While all compositions have exhibited high peelforces, presence of Tris clearly resulted in higher peel forces for thesame FS/ORC ratios, with some showing 2-4 times higher peel force.

TABLE 4 Adhesion Forces of inventive formulations with and without TrisWith no Tris added With Tris added Composition Peel Force Peel ForceTris % by FS/ORC ratio N/m pH N/m pH weight 1:1 26.7 2 72.6 7 20 2:153.8 2 127.5 7 14.3 5:1 41.2 5 235.9 7 7.7 10:1  221.7 5.5 >340 7 4.4(Above the upper limit of measurement)

The analysis of data indicates surprising improvements in adhesive forceor peel force for inventive composition having neutral pH achieved byTris addition. While the force is somewhat lower

Example 7. Characterization of Particles

Referring now to FIG. 9, showing magnified SEM images of 5:1 FS/ORCinventive composition, it is apparent that the components of thecomposition are at least partially integrated, i.e. attached to eachother or coated onto one another, and are not in a simple mechanicalmixture.

Examination of the inventive composition in powder form shows thecomponents well mixed and the biologics were closely attached on the ORCfibers.

Example 8. Hemostasis Testing

An in vivo test of hemostatic efficacy in liver abrasion model using theinventive hemostatic compositions was performed as follows. A liverabrasion model was created by creating an oozing area of 3 cm×3 cm onthe surface of the porcine liver. 0.5 g of the inventive hemostaticcomposition having FS/ORC ratio of 5:1 was applied to cover the oozingarea without any tamponade applied. Hemostasis was achieved in under 2min.

An in vivo test of hemostatic efficacy in liver resection model usingthe inventive hemostatic compositions was performed as follows. A liverresection model was created by using the Pringle maneuver which is asurgical maneuver used in some abdominal operations whereby a largeatraumatic hemostat is applied as a clamp. The Pringle maneuver wasapplied to control bleeding first, then a cut 5 cm long and 5 cm wide ofthe liver tissue was created along the liver edge to expose bile duct.Immediately after, the inventive hemostatic composition powder wasapplied to cover the transection plane, spraying saline simultaneouslyuntil bleeding was stopped. The Pringle clamp was then released toexamine the results. It was observed that hemostasis was achieved andbile leak was prevented after the Pringle clamp was released. Thehemostasis was achieved in 2 min.

Example 9. Method of Manufacturing in High Shear Reactor

According to an embodiment of the present invention, a process of makingpowdered hemostatic product comprises the steps of mixing the individualcomponents as powders with HFE, creating a suspension in HFE (volatilenon-aqueous solvent) in High Shear mixing/shearing reactor, having a lowspeed mixing blade and high-speed shear blade. As mixing is performed,HFE can evaporate through air seals of mixing blades and/or vacuum tube.A small amount of water is introduced into the reactor to aid particlesbinding/aggregation/clumping. The amount of water is sufficient for asmall portion of the biologics to react and form particles, however itis not sufficient for all biologics i.e. thrombin and fibrinogen tofully react so that all fibrinogen is converted into fibrin. The portionof fibrinogen converted into fibrin is from about 0.1% to about 50%,more preferably 1% to 25%, even more preferably 2% to 10%. There isalways an amount of clottable fibrinogen contained in the hemostaticpowders prepared according to the present invention, available forreaction when powder is used on or in a wound.

According to an embodiment of the present invention, a method of makinghemostatic powder, comprising the steps of: Forming a suspension of ORCpowder, thrombin, fibrinogen, lysine, calcium salt in HFE (volatilenon-aqueous solvent), agitating the suspension with high speed shearblade (and optionally continuously mixing the components simultaneouslywith a low speed mixer); Adding a small amount of water to the reactorto aid particles binding/aggregation/clumping; allowing HFE to evaporatefrom the reactor completely; Thus forming the hemostatic powder.

Referring to FIG. 10, a schematic block-diagram of the process of makingthe hemostatic material according to the present invention is shown andcomprises the steps of:

-   -   Preparing dry powders mixture of fibrinogen, thrombin, ORC,        optionally calcium chloride, and optionally buffer compound such        as Tris and/or lysine;    -   Suspending dry powders mixture in a non-aqueous solvent capable        of rapid evaporation under ambient conditions in a High Shear        Mixer reactor    -   Agitating the suspension with high speed shear blade and        optionally continuously mixing the components simultaneously        with a low speed mixer blade    -   Introducing a small amount of water into the reactor to aid        particles binding/aggregation/clumping    -   Allowing the non-aqueous solvent to evaporate from the reactor        drying the resulting hemostatic material    -   Removing the resulting hemostatic material from the reactor and        optionally sieving, thus forming at least partially integrated        ORC fibers, fibrinogen, and thrombin in a form of an aggregated        powder

In one embodiment, Lysine or Tris in a powder form is added to thefibrinogen, thrombin, and ORC mixture for pH adjustment.

According to one aspect of the present invention the inventivehemostatic material comprises substantially uniformly distributed atleast partially integrated ORC fibers, fibrinogen, and thrombin in aform of a powder

According to one aspect of the present invention, the inventivehemostatic material has high uniformity, integration, fastgelling/clotting, and strong adhesion force.

Referring now to FIG. 11, a schematic rendering of a High Shear mixing(HSM) reactor is shown, with container or bowl 5, Impeller 1 for the rawmaterials mixing and suspending (low speed mixer); High speed shearingblade or chopper knife 2; Spray nozzle 3 for the water (binder)additions; Vent 4 for the removal of HFE volatile fluid; Pathways 6 foringress of optional air or gas for purging HFE volatile fluid throughmixing and/or shearing blades seals. Alternatively, a dedicated airintake can be provided (no shown). Alternatively, a vacuum can beconnected to vent 4.

HSM reactors are available commercially. The reactor used in the presentexamples was HSM model Mini-CG, available from Chuangzhi Electrical andMechanical Co., Ltd. (China).

Example 10. Comparative Testing of Hemostatic Powders Made by SprayMethods of Example 1 and HSM Methods of Example 9

The inventors compared the HSM method (Example 9) particles vs.particles made by spraying through a nozzle of HFE suspension (Example1). The test results showed the hemostatic powders made by the HSMmethod had comparable physiochemical properties and functionalperformance like tensile strength and adhesion strength, and hadcomparable hemostatic efficacy in animal study to the powders made bythe spaying method of Example 1.

Comparison of powders of Example 1 formulated with Tris buffer topowders formulated in Example 9 (HSM method) using Lysine as bufferingcompound showed no substantial difference in hemostatic powderperformance.

Advantageously, HSM method of preparing hemostatic powder granulationshas advantages of providing HSM equipment that is a substantially sealedsystem so that powder can be granulated in a sealed environment, and nomaterial is lost during the process. The bioburden could be minimized ascompared to spraying method of Example 1.

Referring to FIG. 12, a hemostatic powder prepared by the HSM Method ofExample 9 is shown.

Example 11. Preparation of Hemostatic Compositions Using HSM Method:Comparison of Various Solvents/Binders

The inventors used the HSM method (Example 9) to manufacture hemostaticpowders using several different solvents/binders. Referring to FIG. 13,a Chart of gel strength in kPa is shown, with the gel formed by thehemostatic powders of the present invention, whereby such powders weremade by using one of four different solvents and/or binders. The gelstrength is shown for several different testing points. First segment ofthe chart shows gel strength for acetone as a volatile suspensionsolvent and polymer RG502 (D, L-lactide-co-glycolide) as a binder. Thesecond segment shows gel strength for ethanol/water combination asvolatile solvent and binder. The third segment shows gel strength foracetone/water combination as volatile solvent and binder. The fourthsegment shows gel strength for HFE/water combination as volatile solventand binder. As can be seen from Chart of FIG. 13, HFE/water combinationshows the highest gel strength.

Referring now to FIG. 14, the results of testing in animal models of thehemostatic powders made by using different solvents/binders arepresented (Hemostasis testing on dermatome liver surface model). As canbe seen from the FIG. 14, combinations of acetone/RG502; ethanol/water;acetone/water all showing bleeding after 3 min after application. To thecontrary, the inventive HFE/water combination shows cessation ofbleeding after only 1 minute.

Example 12. Preparation of Hemostatic Compositions Using HSM Method andPolymer RG502/Acetone

200 g of raw material powder including Fibrinogen powder/Thrombinpowder/ORC powder/Calcium chloride powder and Tris was preparedaccording to the formulation shown in Table 5. The binder/suspensionsolution was prepared from 74.7 g acetone and 8.3 g polymer RG502(D,L-lactide-co-glycolide), with polymer fully dissolved and mixedthoroughly.

TABLE 5 Formulation for Example 12 Raw material Fibrinogen (g) 153.54Thrombin (g) 15.08 Calcium chloride (g) 5.24 ORC (g) 17.40 Tris (g) 8.70Binder Polymer RG502 (g) 8.30 D,L-lactide-co-glycolide Acetone (g) 74.70

The schematic block-diagram of the manufacturing process is shown inFIG. 16. All dry material was transferred into the bowl of HSM, premixedfor 5 minutes using the mixing blade impeller speed at 75 rpm, highshear blade chopper speed at 1000 rpm. The binder/suspension solution(RG502/acetone) was then sprayed onto the dry materials by a Peristalticpump and a spray nozzle, with the feed flowrate 60 g/min. The solutionof polymer/acetone starts binding the raw material particles to formpowder.

The Impeller speed was then increased to 150 rpm and the chopper speedincreased to 3000 rpm, continuing the post granulation process for 5mins.

After granulation, a sieve (the pore size is 1.7 mm) was used to sievethe materials and collect the resulting powder under the sieve. Thepowder was transferred to vacuum drying box and dried for 1 hour. Twosieves (pore size 106 and 425 μm) were then used to sieve the product.The final powder fraction was collected between the two sieves.

Example 13. Preparation of Hemostatic Compositions Using HSM Method andWater/Acetone

200 g of raw material powder including Fibrinogen powder/Thrombinpowder/ORC powder/Calcium chloride powder and Tris was preparedaccording to the formulation shown in Table 6. The binder/suspensionsolution was prepared from 68 g acetone and 17 g of purified water,mixed thoroughly.

TABLE 6 Formulation for Example 13 Raw material Fibrinogen (g) 153.54Thrombin (g) 15.08 Calcium chloride (g) 5.24 ORC (g) 17.40 Tris (g) 8.70Binder Water (g) 17.00 Acetone (g) 68.00

The schematic block-diagram of the manufacturing process is shown inFIG. 18. All dry material was transferred into the bowl of HSM, premixedfor 5 minutes using the mixing blade impeller speed at 75 rpm, highshear blade chopper speed at 1000 rpm. The binder/suspension solution(water/acetone) was then sprayed onto the dry materials by a Peristalticpump and a spray nozzle, with the feed flowrate 60 g/min. The solutionstarts binding the raw material particles to form powder.

The Impeller speed was then increased to 120 rpm and the chopper speedincreased to 3000 rpm, continuing the post granulation process for 3mins.

After granulation, a sieve (the pore size is 1.7 mm) was used to sievethe materials and collect the resulting powder under the sieve. Thepowder was transferred to vacuum drying box and dried for 1 hour undervacuum. Two sieves (pore size 106 and 425 μm) were then used to sievethe product. The final powder fraction was collected between the twosieves

Example 14. Preparation of Hemostatic Compositions Using HSM Method and96% Ethanol

150 g of raw material powder including Fibrinogen powder/Thrombinpowder/ORC powder/Calcium chloride powder and Tris was preparedaccording to the formulation shown in Table 7. The binder/suspensionsolution was prepared from 60 g of 96% ethanol.

TABLE 7 Formulation for Example 14 Raw material Fibrinogen (g) 115.16Thrombin (g) 11.31 Calcium chloride (g) 3.93 ORC (g) 13.05 Tris (g) 6.53Binder 96% Ethanol (g) 60.00

The schematic block-diagram of the manufacturing process is shown inFIG. 20. All dry material was transferred into the bowl of HSM, premixedfor 3 minutes using the mixing blade impeller speed at 75 rpm, highshear blade chopper speed at 1000 rpm. The binder/suspension solution(96% ethanol) was then sprayed onto the dry materials by a Peristalticpump and a spray nozzle, with the feed flowrate 4 g/min. The solutionstarts binding the raw material particles to form powder.

The Impeller speed was then increased to 120 rpm and the chopper speedincreased to 1500 rpm, continuing the post granulation process for 3mins.

After granulation, a sieve (the pore size 710 μm) was used to sieve thematerials and collect the resulting powder under the sieve. The powderwas transferred to tray oven for drying and dried at 45° C. for 0.5 hr.

Two sieves (pore size 100 and 315 μm) were then used to sieve theproduct. The final powder fraction was collected between the two sieves

Example 15. Preparation of Hemostatic Compositions Using HSM Method andHFE/Water

100 g of raw material powder including Fibrinogen powder/Thrombinpowder/ORC powder/Calcium chloride powder and Tris was preparedaccording to the formulation shown in Table 8. 500 g of HFE7100 wasutilized.

TABLE 8 Formulation for Example 15 Raw material Fibrinogen (g) 76.77Thrombin (g) 7.54 Calcium chloride (g) 2.62 ORC (g) 8.70 Tris (g) 4.35Suspending agent HFE 7100 (g) 500.00 Binder Water (g) 9.00

The schematic block-diagram of the manufacturing process is shown inFIG. 22. All dry material was transferred into the bowl of HSM and 500 gof HFE7100 added. The composition was premixed for 3 minutes to form asuspension, using the mixing blade impeller speed at 200 rpm. 9 g ofwater was then sprayed into the suspension by a Peristaltic pump and aspray nozzle, with the feed flowrate of 4.5 g/min. The solution startsbinding the raw material particles to form powder.

The Impeller speed was then adjusted to 100-300 rpm and the chopperspeed set at 150-1000 rpm, continuing the granulation process for 10mins. Sealing pressure of impeller and chopper was adjusted to 0.02 MPato blow off HFE and dry the composition.

After granulation, a sieve (the pore size 710 μm) was used to sieve thematerials and collect the resulting powder under the sieve. The powderwas transferred vacuum box for drying and dried for 12-24 hours atvacuum from 0-10 Pa.

Two sieves (pore size 106 and 355 μm) were then used to sieve theproduct. The final powder fraction was collected between the two sieves.

Example 16. Comparison of Hemostatic Powder Formulations Obtained inExamples 12, 13, 14, 15

Hemostatic powders obtained as described in Examples 12, 13, 14, 15 werecompared for their performance. Water content was tested by Karl Fischermethod; Thrombin potency was tested based on clotting time; Clottableprotein was tested by quantifying fibrinogen; Particle size was testedby Laser particle size analyzer; Density was measured as Bulk density;Gel strength was measured as Tensile strength test. The results of thetesting are shown in Table 9 and in FIGS. 19, 20.

TABLE 9 Properties of hemostatic powders obtained with varioussolvents/binders Particle Water Thrombin Clottable size Gel contentpotency Protein Density D50 strength (%) (IU/mg) (mg/g) (g/ml) (μm)(Kpa) Example 2.32 1.78 290.11 0.33 181.97 61.13 12 Example 2.44 2.19324.55 0.38 201.57 55.35 13 Example 3.87 2.03 284.08 0.34 71.98 55.00 14Example 2.80 1.80 326.76 0.29 57.66 121.15 15

Advantageously, the volatile properties of HFE potentially increase theporosity of final hemostatic powder product, which will lead to adecrease in the density of the final product, and speeds up thedissolution or re-dispersion time. The density of the powders of Example15 is lowest, while the strength of formed gel is highest vs. othermethods. The hemostatic performance was also better as was shown in FIG.14.

Example 17. Testing of the Inventive Hemostatic Powder in Lung SealingModel

Two studies were conducted to test the hemostatic powders of the presentinvention for sealing function on lung leak model. Both studies showedat 30 cm H2O pressure, the present powder formed gel that was tightlyadhering to the lung tissue and sealed the leak very well. When thepressure was 40 cm H2O, the gel started to delaminate, but still couldseal the leak.

FIG. 21 shows Lung leak model with 1.2 cm long incision;

FIG. 22 shows the inventive hemostatic powder forming a gel (counted for2 min) on the deflated lung;

FIG. 23 shows the inventive hemostatic powder forming a gel that sealsthe lung leak at 30 cm H2O;

FIG. 24 shows Lung leak model with 1 cm long incision;

FIG. 25 shows the inventive hemostatic powder forming a gel that sealsthe lung leak at 30 cm H2O;

FIG. 26 shows the inventive hemostatic powder forming a gel partiallydelaminated at 40 cm H2O pressure (arrow indicates the delaminationarea).

Advantageously, the inventive hemostatic powder is shown to be able toseal lung leaks.

I/We claim:
 1. A method of forming a powdered hemostatic composition, comprising the steps of: a) forming a suspension of a mixture comprising particles of fibrinogen, thrombin, ORC fibers in a non-aqueous low boiling solvent; b) agitating and shearing said suspension in a high shear mixing reactor c) adding water to allow particles to agglomerate; d) allowing the non-aqueous solvent to evaporate; e) drying and sieving the composition; and thus forming the powdered hemostatic composition.
 2. The method of claim 1, wherein said non-aqueous low boiling solvent comprises Hydrofluoroether C₄F₉OCH₃.
 3. The method of claim 2, wherein said non-aqueous low boiling solvent comprises HFE7100.
 4. The method of claim 1, wherein said suspension further comprises Lysine or Tris.
 5. The method of claim 1, wherein said suspension further comprises calcium chloride.
 6. The method of claim 1, wherein said water is added in a quantity preventing full clotting of fibrinogen.
 7. The method of claim 1, wherein said water is added in a quantity resulting in less than 50% of fibrinogen conversion into fibrin.
 8. A method of treating a wound by applying the hemostatic composition formed by method of claim 1 onto and/or into the wound.
 9. A method of treating an air leak in a lung by applying the hemostatic composition formed by method of claim 1 onto and/or into the lung. 